Carbamate Stereoisomer

ABSTRACT

The compound of formula (I) 
     
       
         
         
             
             
         
       
     
     is a water-stable, long acting β 2 -selective adrenoceptor agonist useful as a bronchodilator in the treatment of bronchoconstriction associated with reversible obstructive airways diseases and the like.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.60/966,391 filed Aug. 28, 2007, the entire disclosure of which is hereinincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a novel carbamate stereoisomer, to aprocess for preparing the carbamate stereoisomer, to a pharmaceuticalcomposition comprising the carbamate stereoisomer and to the use of thecarbamate stereoisomer in therapy, in particular in the treatment ofbronchoconstriction associated with reversible obstructive airwaysdiseases including but not limited to asthma, cystic fibrosis andchronic obstructive pulmonary disease, including chronic bronchitis andemphysema.

BACKGROUND OF THE INVENTION

Patients suffering from bronchoconstriction associated with reversibleobstructive airways diseases are generally treated using abronchodilator, to relax the bronchial smooth muscle.

Bronchodilators in use today generally fall into two classes, theβ₂-selective adrenoceptor agonists, such as albuterol (salbutamol),salmeterol and formoterol, and the muscarinic receptor antagonists, suchas ipratropium and tiatropium.

β₂-Selective adrenoceptor agonists may cause adverse effects, and thesemay in part be due to activation of the β₁-adrenoceptor. The selectivityof an agonist for the β₂-adrenoceptor receptor is therefore veryimportant, because it limits the dose that can be given and so affectsthe magnitude of bronchodilations and the frequency of dosing.

A long duration of action is important to patients, not only to minimizethe time spent taking the drug, but also to avoid having to take thedrug during inconvenient times, for example at work, school or duringthe night. Some of the more recent β₂-selective adrenoceptor agonists,in particular salmeterol and formoterol, have a long duration of action,typically about 12 hours. Formoterol has a particular advantage that italso has a fast onset of action. However, formoterol is extremelypotent, which makes it very difficult to formulate, especially foradministration using a metered dose inhaler in a manner that results inuniform drug delivery via aerosol dose after dose (i.e., dose contentuniformity). Furthermore, it is unstable in aqueous solution, whichmeans that solutions for administration using a nebuliser have to bekept refrigerated for a majority of their post-manufacture shelf life.

Formoterol is one of a group of α-aminomethylbenzyl alcohol derivativesfor which patent applications were filed during the early nineteenseventies, for example U.S. Pat. No. 3,994,974. The invention of thiscompound built on earlier work by others, such as described in U.S. Pat.No. 3,657,319 (equivalent to BE 765,986, cited in U.S. Pat. No.3,994,974). Perhaps because of the difficulties associated withformulating the compound, it took a long time to be commercialized. Thecompound contains two chiral centers, and hence is capable of existingand being isolated in four stereoisomeric forms. The compound wasfirstly commercialized as a racemic mixture of the active (R,R)- andinactive (S,S)-isomers, in a dry powder formulation, then more recentlyas the active (R,R)-isomer in a nebuliser solution. It is also known,for example from U.S. Pat. No. 6,303,145, that the (S,R) isomer offormoterol is active. However, like the (R,R)-isomer, this compound isunstable at ambient temperature in aqueous solution and hence nebulisersolutions would need to be stored refrigerated.

SUMMARY OF THE INVENTION

Surprisingly, it has now been found that by replacing the methoxy groupin (S,R)-formoterol with a hydroxy group, and the formyl hydrogen atomwith a methoxy group, an isomer having a particularly attractivecombination of properties has been obtained.

According to one aspect, therefore, the present invention provides acompound of formula (I)

or a pharmaceutically acceptable salt thereof.

The compound of formula (I) may also be referred to by the chemical namemethyl[2-hydroxy-5-[1-hydroxy-2-[[(2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-phenyl]carbamate, which is indexed in Chemical Abstracts as carbamic acid,[2-hydroxy-5-[(1S)-1-hydroxy-2-[[(1R)-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-phenyl]-,methyl ester.

The isomer of formula (I) has been found to possess particularlyadvantageous properties. In particular, it possesses good, but not veryhigh affinity for the β2-adrenoceptor, high selectivity for the β2- overthe β₁-adrenoceptor, a long duration of action and good stability inaqueous solution at ambient temperature.

DETAILED DESCRIPTION OF THE INVENTION

It will be appreciated that the compound provided by the presentinvention is an isomer. This isomer may exist and be isolated inenantiomerically pure form, or in admixture with one or more of itsother isomers. The present invention provides the isomer in any mixtureof isomers other than a racemic mixture, which is described in Example 6of U.S. Pat. No. 3,657,319. In certain embodiments, the isomer issubstantially free of the (R,R)-enantiomer, which can exhibit adifferent potency, resulting in significant variations in the potency ofadmixtures. It may exist as a 1:1 diastereomeric mixture with the(R,S)-isomer, but is most preferably enantiomerically pure (i.e.substantially free of all other isomers). For example, the isomer maycomprise at least 50% by weight of all carbamic acid,[2-hydroxy-5-[1-hydroxy-2-[[(2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]phenyl]-,methyl ester present, preferably at least 75%, such as at least 90%, atleast 95% or at least 99%.

As used herein, the term “pharmaceutically acceptable salt” refers to asalt prepared from a pharmaceutically acceptable, relatively non-toxicacid, including inorganic acids and organic acids. Suitable acidsinclude acetic, benzenesulfonic, benzoic, camphorsulfonic, carbonic,citric, dihydrogenphosphoric, ethenesulfonic, fumaric, galactunoric,gluconic, glucuronic, glutamic, hydrobromic, hydrochloric, hydriodic,isobutyric, isethionic, lactic, maleic, malic, malonic, mandelic,methanesulfonic, monohydrogencarbonic, monohydrogenphosphoric,monohydrogensulfuric, mucic, nitric, pamoic, pantothenic, phosphoric,phthalic, propionic, suberic, succinic, sulfuric, tartaric,toluenesulfonic, including p-toluenesulfonic m-toluenesulfonic ando-toluenesulfonic acids, and the like (see, e.g., Berge et al., J.Pharm. Sci., 66:1-19 (1977); Stahl and Wermuth, Handbook ofPharmaceutical Salts, Wiley VCH, (2002)). Also included are salts ofother relatively non-toxic compounds that possess acidic character,including amino acids, such as arginine and the like, and othercompounds, such as aspirin, ibuprofen, saccharin, and the like. Acidaddition salts can be obtained by contacting the neutral form of suchcompounds with a sufficient amount of the desired acid, either neat orin a suitable inert solvent. As solids, salts can exist in crystallineor amorphous modifications. An example of an acid addition salt is theD-tartrate salt.

The compounds of the present invention may also be prepared indeuterated form, i.e., in which one or more hydrogen atoms, for exampleon the methoxycarbonyl group, are replaced with deuterium.

It is also contemplated that the methyl group in the methoxycarbonylgroup in the compound of formula (I) may be replaced with a fluoromethylgroup (i.e. a group in which one, two or three of the methyl hydrogenatoms is replaced with a fluorine atom). Such compounds may be preparedby a process analogous to that described herein for the preparation ofthe carbamate isomer.

The carbamate isomer and its pharmaceutically acceptable salts can beprepared by a process, which comprises reacting a compound of generalformula (II)

in which P¹ represents a hydrogen atom or a hydroxyl protecting group,with a compound of general formula (III)

in which P² represents a hydrogen atom or a hydroxyl protecting groupand P³ represents a benzylic amine protecting group, to afford acompound of general formula (IV)

or a salt thereof, followed by removing any protecting groups P¹, P² andP³ and, if desired, forming a pharmaceutically acceptable acid additionsalt. The protecting groups may be any suitable protecting group, forexample as described in Green et al., “Protective Groups in OrganicChemistry,” (Wiley, 2^(nd) ed. 1991). Examples of hydroxyl protectinggroups include aralkyl groups, such as benzyl, and trialkylsilyl groups,such as t-butyl-dimethylsilyl (TBDMS). Examples of a benzylic amineprotecting group are benzyl groups optionally substituted on the benzenering by one or more, for example 1, 2 or 3 optional substituents, forexample selected from halo, (1-4C) alkyl and (1-4C)alkoxy; for exampleunsubstituted benzyl.

The reaction between the compounds of formula (II) and (III) isconveniently performed by melting the two compounds together, forexample by heating in the range of from 50 to 130° C., such as about 75°C.

Any protecting groups represented by P¹, P² and P³ may be removed usinga conventional procedure. For example, a benzyl group can be removed bycatalytic hydrogenation in the presence of palladium on carbon, and atrialkylsilyl group by treatment with tetrabutylammonium fluoride.

Compounds of formula (II) can be prepared by reacting a compound offormula (V)

in which Z represents a leaving atom or group, such as a bromine atom,with a base, for example an alkali metal carbonate such as potassiumcarbonate.

Compounds of formula (V) can be prepared by stereoselective reduction ofa compound of formula (VI)

using, for example, borane in the presence of a chiral auxiliary, suchas (1S,2R)-1-amino-2-indanol, followed by reduction of the nitro groupto an amino group and acylation of the resultant amino group, forexample using dimethyl carbonate.

Compounds of general formula (III) can be prepared by reacting acompound of general formula (VII)

with boron tribromide, to afford a compound of formula (VIII)

The hydroxyl group may then be protected, for example by reaction with atrialkylsilyl halide, such as t-butyldimethylsilyl chloride.

It will be appreciated that the percentage by weight comprised by thecompound of formula (I) of all carbamic acid,[2-hydroxy-5-[1-hydroxy-2-[[(2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]phenyl]-,methyl ester present in the final product of the process will dependupon the enantiomeric purity of the starting materials used and anyenantiomeric purification steps taken, such as chiral liquidchromatography.

The intermediates of general formula (IV) are believed to be novel andare provided as a further aspect of the present invention.

According to another aspect, therefore, the present invention provides apharmaceutical composition, which comprises a compound of formula (I) ora pharmaceutically acceptable salt thereof, as described herein,together with a pharmaceutically acceptable carrier.

The pharmaceutical composition according to the invention may be adaptedfor administration to patients by any convenient route, such as by oral,mucosal (e.g. nasal, sublingual, vaginal, buccal or rectal), parenteralor transdermal administration. It may be in the form of, for example, asolution, suspension, powder, tablet, aerosol formulation, lozenge,suppository, emulsion, hard or soft gelatin capsule or syrup. Thecompound of formula (I) may be dissolved in the carrier, diluted by thecarrier or supported by the carrier. Thus the carrier may be a supportfor the compound of formula (I), such as a capsule, sachet, paper orother pharmaceutical container.

In one embodiment, the pharmaceutical composition is an aqueous solutionadapted for administration using a nebuliser. The aqueous formulationmay be isotonic and buffered at an optimal pH for stability. The aqueousformulation for nebulization could also be a suspension of nanoparticlesor a micronized suspension of free base or an insoluble salt or acyclodextrin adduct.

In another embodiment, the pharmaceutical composition is an aerosolformulation adapted for administration using a metered dose inhaler, theaerosol formulation comprising the acetamide isomer in crystalline formand a propellant or in solution with an appropriate propellant,combination of propellants or combination of propellant(s) and anacceptable co-solvent or other solubilizing agent.

The propellant may be any suitable propellant used in aerosolformulations, for example, a hydrofluoroalkane (HFA), such as1,1,1,2-tetrafluoroethane (HFA134) or 1,1,1,2,3,3,3-heptafluoropropane(HFA227) or a combination of propellants. HFA134 is preferred. Thepropellant may comprise at least 90% by weight of the aerosolformulation, which may also include, inter alia, inert gases to aide inaerosol formation.

The aerosol formulation may further comprise a surfactant. Thesurfactant serves to stabilize and disperse the carbamate isomer in asuspension, and may also serve as a valve lubricant in the metered doseinhaler. It may be any suitable surfactant used in aerosol formulations.Examples of surfactants used in aerosol formulations are described inU.S. Pat. No. 5,225,183, which is hereby incorporated by reference. Apreferred surfactant is oleic acid. The surfactant, when present, maygenerally be present in an amount of from 1:100 to 1:10 surfactant:carbamate isomer, preferably about 1:20.

The aerosol formulation may further comprise a co-solvent. A function ofthe co-solvent in the aerosol formulation is to facilitate dissolutionof the surfactant, which may have poor solubility in the propellant. Itmay be any suitable carrier used in aerosol formulations. A co-solventsuch as glycerol or ethanol may be used. A preferred co-solvent isethanol, especially dehydrated ethanol. The content of ethanol mayconveniently be up to 30% by weight of the aerosol formulation, such asfrom 2 to 6%.

Metered dose inhalers typically comprise a canister containing anaerosol formulation, a metering valve, a valve stem and an actuatorwhich accepts the valve stem. In use, a patient depresses the canisterinto the actuator and inhales, causing a dose of the formulation to beadministered and taken into the patient's lungs.

According to a further aspect, therefore, the present invention providesa metered dose inhaler comprising a canister containing an aerosolformulation as described herein, a metering valve and an actuator.

Preferably the interior surface of the canister is coated, for examplewith a protective polymer, or otherwise treated to minimize chemical orphysical interaction between the formulation and the canister. Theinhaler preferably has an aperture with a diameter in the range of from0.2 to 0.60 mm.

In yet another embodiment, the pharmaceutical composition is in the formof a dry powder suitable for inhalation or insufflation. The compositionmay comprise carbamate isomer crystals alone (e.g. having a mass medianaerodynamic diameter of from 1 to 10 microns, preferably from 2 to 7microns), or carbamate isomer blended, co-precipitated, co-crystallizedor spray dried together with a suitable pharmaceutically acceptablecarrier or carriers. Suitable pharmaceutically acceptable carriersinclude, without limitation, solvates of one or more natural orsynthetic carbohydrates, such as a monosaccharides, disaccharides,trisaccharides, oligosaccharides, polysaccharides, polyols, amino acidsand proteins, and/or in the form of their pharmaceutically acceptableesters, acetals, or salts (where such derivatives exist). The carrier ispreferably lactose, more preferably lactose monohydrate. The dry powdercomposition may be presented in unit dosage form in, for example,capsules or cartridges of e.g. gelatin, or blister packs from which thepowder may be administered with the aid of an inhaler or insufflator.The dry powder composition may be presented in multi dose form meteredwith the aid of an inhaler or insufflator, or pre-metered into discretedoses within the device for serial administrations.

Conveniently, dry powder formulations are administered using multidosedry powder inhalers.

The present invention therefore also provides a multidose dry powderinhaler, comprising a dry powder reservoir containing a dry powderaerosol formulation of carbamate isomer as described hereinabove, and ametering chamber.

The compound of formula (I) according to the present invention may beco-administered with one of more other active ingredients, for exampleselected from steroids, such as beclomethasone, triamcinolone,funisolide, mometasone, budesonide or fluticasone, muscarinic receptorantagonists, such as ipratropium, tiatropium, or glycopyrrolate.Accordingly, in one embodiment, the pharmaceutical composition inaccordance with the present invention may further comprise a steroidand/or a muscarinic receptor antagonist and/or a controller agent orbronchodilator with a novel mechanism.

In another embodiment, the pharmaceutical composition in accordance withthe present invention may further comprise anti-inflammatory agents suchas inhibitors of tumor necrosis factor alpha (TNFα), dipeptidylpeptidase IV, and antibodies to pro-inflammatory interleukins such asIL4 and IL13.

In another embodiment, the pharmaceutical composition in accordance withthe present invention may further comprise mucolytic agents such ascromoglycate, acetylcysteine, arginine, or 2-mercaptoethanesulphonate.

According to another aspect, the present invention provides a method oftreating bronchoconstrictive disease, which comprises administering to apatient in need of treatment an effective amount of a compound offormula (I) or a pharmaceutically acceptable salt thereof.

The bronchoconstrictive disease may be, for example, chronic obstructivepulmonary disease (such as emphysema or bronchitis), cystic fibrosis, orasthma.

The patient may be a human or a non-human mammal, such as a dog, cat,horse, cow, sheep or pig. Preferably, the patient is a human.

The amount of compound administered will depend upon many factors, suchas the species, weight and age of the patient, and the severity of thecondition to be treated. For example, a dose administered to a human maycontain from 75 to 5,000 μg of the carbamate isomer (calculated as thefree base). The dose may be administered, for example, once or twice perday.

According to another aspect, the present invention provides a compoundof formula (I) or a pharmaceutically acceptable salt thereof, for use intherapy.

According to yet another aspect, the present invention provides the useof a compound of formula (I) or a pharmaceutically acceptable saltthereof in the manufacture of a medicament for the treatment of chronicobstructive pulmonary disease.

According to a still further aspect, the present invention provides apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier for the treatment of chronic obstructive pulmonarydisease, or for use as a bronchodilator.

Although the foregoing invention has been described in some detail forpurposes of illustration, it will be readily apparent to one skilled inthe art that changes and modifications may be made without departingfrom the scope of the invention described herein.

EXAMPLES

The following Examples illustrate the invention.

THF refers to tetrahydrofuran, EtOAc refers to ethyl acetate and Et₂Orefers to diethyl ether.

Example 1

Carbamic acid,[2-hydroxy-5-[(1S)-1-hydroxy-2-[[(1R)-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-phenyl]-,Methyl Ester

Step A) (1S)-1-(3-nitro-4-benzyloxyphenyl)-2-bromoethan-1-ol

A cold (5° C.) solution of (1S,2R)-1-amino-2-indanol (400 mg, 2.68 mmol)in THF (160 mL) was added dropwise to a cold (0° C.) solution ofborane-diethylaniline complex (7.0 g, 43 mmol) in THF (20 mL). Aftercomplete addition, the resulting solution was stirred at (0° C.) for 30min then 2-bromo-4′-benzyloxy-3′-nitroacetophenone (20.0 g, 57.1 mmol)was added in three portions over a 30 min period. The resulting solutionwas stirred at <5° C. for 1 h, quenched by dropwise addition of acetone(17 mL) then allowed to warm to ambient temperature overnight. Thereaction mixture was concentrated in vacuo to a residue, which wasdissolved in toluene (100 mL) and washed in succession with 10% H₂SO₄(2×45 mL), H₂O(2×45 mL) and sat. brine (1×40 mL). The organic layer wasdried over MgSO₄, clarified then concentrated in vacuo to a volume of˜40 mL. Heptane (45 mL) was slowly added to give a thick slurry. Thesolid was collected on a filter and washed with heptane (2×5 mL). Thismaterial was dissolved in warm toluene (˜50 mL), the solution wasclarified then diluted with heptane (50 mL). The resulting mixture wasstirred for 30 min, the solids were collected, washed with heptane (2×5mL) then dried to constant weight in vacuo to give 18.9 g (94.0%) of thetitle compound.

Step B) (1S)-1-(3-amino-4-benzyloxyphenyl)-2-bromoethan-1-ol

A solution of the product of Step A) (18.7 g, 53.1 mmol) in toluene (40mL) and THF (40 mL) was added to a Parr shaker bottle containing Pt₂O(370 mg). This mixture was shaken under H₂ (50 psi, 344.74 kpa) untilthe reaction was complete (18 h). The catalyst was removed byfiltration, and the filtrate was concentrated to an oil. Columnchromatography (1 kg silica gel packed in and eluted with CH₂Cl₂/MeOH,19:1) gave 11.9 g (69.6%) of the title compound.

Step C)(1S)-1-(3-methoxycarbonylamino-4-benzyloxyphenyl)-2-bromoethan-1-ol

A solution of the product of Step B) (11.7 g, 36.3 mmol) in pyridine(117 mL) was stirred at ambient temperature for 15 min. Dimethyldicarbonate (4.9 g, 37 mmol) was added in one portion, and the resultingsolution was stirred at ambient temperature for 30 min followed by 4 hat 40° C. Additional dimethyl dicarbonate (1.3 g, 9.7 mmol) was addedand stirring continued at 40° C. for 2 h. The reaction solution wasconcentrated in vacuo to an oil that was partitioned between CH₂Cl₂ (470mL) and 10% aq. HCl (120 mL). The aqueous layer was extracted withCH₂Cl₂ (1×120 mL). The combined organic layer was washed in successionwith H₂O (1×350 mL) and brine (1×350 mL), dried over MgSO₄, clarifiedthen concentrated in vacuo to a thick slurry. The mixture was dilutedwith hexanes (120 mL) and stirred for 30 min. The resulting solid wascollected on a filter, washed with hexanes (2×60 mL) then dried toconstant weight in vacuo to give ˜10 g of a white solid. This materialwas further purified by trituration in Et₂O (60 mL). The solid wascollected on a filter, washed with Et₂O (2×10 mL) then dried to constantweight in vacuo to give 8.5 g of a partially purified product. Thismaterial was further purified by silica gel chromatography (800 gcolumn, packed in and eluted with CH₂Cl₂/MeOH, (19:1) to give 7.2 g(52%) of the title compound suitable for further transformation.

Step D) (1S)-1-(3-methoxycarbonylamino-4-benzyloxyphenyl)-epoxyethane

A suspension of the product of Step C) (3.5 g, 9.2 mmol) and potassiumcarbonate (1.7 g, 12 mmol) in methanol (30 mL) and THF (30 mL) wasstirred at ambient temperature for 2.5 h, then concentrated in vacuo toa residue. The residue was triturated with CH₂Cl₂ (35 mL), and theresultant solid was discarded after washing with CH₂Cl₂ (2×8 mL). Thecombined CH₂Cl₂ extracts were concentrated to a residue that wastriturated in hexanes/ether (1:9) (50 mL). The solid was collected on afilter, washed with hexanes (2×15 mL) then dried to constant weight invacuo to give 1.4 g (51%) of the epoxide. An identical run using 3.7 g(9.7 mmol) of starting material was carried out to give 1.7 g (59%) offurther product for a total of 3.1 g of the title epoxide suitable forfurther transformation.

Step E) [(1R)-N-Benzyl-2-(4-hydroxyphenyl)-1-methylethyl]-amine

To a solution of [(1R)-N-Benzyl-2-(4-methoxyphenyl)-1-methylethyl]amine(5.30 g, 20.8 mmol) in CH₂Cl₂ (25 mL) was added a solution of BBr₃ inCH₂Cl₂ (25.0 mL, 1.OM, 25.0 mmol) slowly over 0.5 h. After the addition,the mixture was stirred at ambient temperature for 22 h. Water (125 mL)was added, followed by the addition of 2.5M aq. NaOH (15 mL) to pH 6.The mixture was extracted with EtOAc (4×200 mL), and the organic layerwas dried (Na₂SO₄) and concentrated. The residue (3.9 g) was trituratedwith CH₂Cl₂ (120 mL) and then concentrated to dryness to give the titlecompound (3.8 g, 76%).

Step F)[(1R)-N-Benzyl-2-(4-t-butyldimethylsilyloxyphenyl)-1-methylethyl]amine

A solution of the product of Step E) (3.20 g, 13.3 mmol),tert-butyldimethylsilyl chloride (3.59 g, 23.8 mmol), and imidazole(2.86 g, 42.0 mmol) in DMF (30.0 mL) was stirred at ambient temperaturefor 18 h. The mixture was concentrated to dryness, and the residue waspartitioned between EtOAc (200 mL) and sat. aq. NaHCO₃ (200 mL). Theaqueous layer was separated and again extracted with EtOAc (100 mL). Thecombined organic layer was washed with brine (100 mL), dried withNa₂SO₄, filtered and concentrated to give an oil. The oil waschromatographed on silica gel (100 g, eluted with 1:1 EtOAc:hexanes) togive the title compound (4.0 g, 85%) as a tan oil.

Step G) Carbamic acid,[2-benzyloxy-5-[(1S)-1-hydroxy-2-[[(1R)-2-(4-t-butyldimethylsilyloxyphenyl)-1-ethylethyl]-N-benzylamino]ethyl]phenyl]-, Methyl Ester

A mixture of the products of Step D) (1.26 g, 4.21 mmol) and Step F)(1.50 g, 4.22 mmol) was heated slowly to 75° C. without any solvent. Theresulting mixture was heated at this temperature for a total of threedays at which time TLC evaluation (hexanes/EtOAc, 3:1) indicatedcomplete loss of starting epoxide. After cooling to room temperature therather complex reaction mixture was purified by chromatography two timeson a column of silica gel (100 g) packed in and eluted withhexanes/EtOAc (3:1). A total of 2.9 g of the epoxide was reacted andpurified in a similar manner to give 2.6 g (41%) of the title compoundas a yellow oil. This material was suitable for further transformation.

Step H) Carbamic acid,[2-benzyloxy-5-[(1S)-1-hydroxy-2-[[(1R)-2-(4-hydroxyphenyl)-1-methylethyl]-N-benzylamino]-ethyl]phenyl]-,Methyl Ester

To a solution of the product of Step G) (1.7 g, 2.6 mmol) in anhydrousTHF (17 mL) cooled with an ice water bath was added tetrabutylammoniumfluoride solution (5.3 mL of 1.0 M solution in THF, 5.3 mmol) whilestirring under argon. The reaction mixture was stirred at ambienttemperature for 1 h, and TLC evaluation (EtOAc/hexanes, 1:1) indicated acomplete reaction. The reaction mixture was diluted with EtOAc (200 mL)and washed twice with water (2×120 mL), then dried (MgSO₄), filtered,and concentrated to give crude product as a yellow oil. The crudematerial was purified by silica gel column chromatography (2×200 gcolumns), packed in and eluted with 1:1 EtOAc/hexanes [the crude productwas loaded onto the column as a solution in CH₂Cl₂]. Fractionscontaining the purified product were combined and concentrated to givean oil. A total of 2.6 g of starting material was deblocked to give atotal of 1.2 g (56%) of the title compound after purification.

Step I) Carbamic acid,[2-hydroxy-5-[(1S)-1-hydroxy-2-[[(1R)-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]phenyl]-,Methyl Ester

A mixture of the product of Step H) (300 mg, 0.555 mmol) 10% Pd/C (400mg, dry material) and EtOH (30 mL) was hydrogenated (ambienttemperature) at 50 psi (344.74 kpa) for 22 h. The catalyst was filteredoff, and the filtrate was spin evaporated to afford the title compoundas an oil (200 mg). [check this—is this the right experimental toinclude?]

Step J) Carbamic acid,[2-hydroxy-5-[(1S)-1-hydroxy-2-[[(1R)-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-phenyl]-,Methyl Ester, (2S,3S)-2,3-dihydroxybutanedioate (1:1) (Salt)

480 mg of product prepared by the method of Step I) was dissolved in 0.9mL of i-PrOH. A solution of 151 mg (1.00 mmol) of D-tartaric acid in 0.3mL of H₂O was added. The reaction mixture was stored at 5° C. for 16 h.Crystallization did not occur. The mixture was subjected to spinevaporation to remove volatiles to produce a brown gum. Attemptedtriturations using acetone then iPrOH (10 mL each) were unsuccessful.This material (gum) was diluted with isopropyl ether (15 mL). Afterstirring for 3 h, a light brown, free flowing solid was formed. Thesolid was collected on a funnel, washed with 5 mL of isopropyl etherthen vacuum dried at room temperature for 60 h to constant weight togive 480 mg of the title compound. MS m/z:[M+H⁺] 361. ¹H NMR spectrumconsistent with the assigned structure.

β₁ and β2 radioligand binding assays

The affinity of a test compound for adrenergic β₁ and β₂ receptors isinvestigated by evaluating the ability of the compound to displacespecific binding of [¹²⁵I]-cyanopidolol or [³H]-CGP-12177 at humanrecombinant β₁ and β₂ receptors, respectively (expressed in CHO cells).The IC₅₀ is defined as the concentration that inhibits 50% of specificbinding of the radioligand. The K_(i) is calculated from the IC₅₀ andthe known K_(D) of the radioligand (Cheng and Prusoff's equation).

In this test, the compound of Example I was found to afford a K_(i)of >10 μM with only 44% inhibition of specific binding at aconcentration of 20 μM for the β₁ receptor and 0.99 μM for the β₂receptor. The β₁/β₂ binding ratio was found to be >10.

By way of comparison, the values found for arformoterol and the (S,R)isomer of formoterol were 0.155 μM (β₁), 0.004 μM (β₂) and 41(β₁/β₂),and 2.50 μM (β₁), 0.075 μM (β₂) and 33(β₁/β₂), respectively.

Intrinsic Activity Assessment (β₂)

The intrinsic activity of a test compound is assessed by evaluating itsability to increase cAMP production from human recombinant β₂ receptorsexpressed in CHO cells. Data are expressed as % response relative to aprocaterol-induced cAMP increase.

The compound of Example 1 was found to have an intrinsic activity of79%.

By way of comparison, arformoterol and (S,R)-formoterol were found tohave intrinsic activities of 98% and 91% respectively.

β₁ and β₂ Adrenergic Activity (Functional)

Functional agonism at adrenergic β₁ receptors is demonstrated by apositive chronotropic effect in isolated right atria from Dunkin HartleyGuinea pigs. The concentration that gives 50% maximal effect is theEC₅₀.

Functional agonism at adrenergic β₂ receptors is demonstrated byrelaxation of the spontaneous tone of isolated trachea from DunkinHartley Guinea pigs. The concentration that gives 50% maximal effect isthe EC₅₀.

In these tests, an EC₅₀ could not be determined for the compound ofExample 1 for the β₁ functional assay as only a 20% increase in heartrate was seen at a concentration of 30 μM. However, the compound ofExample 1 was found to have an EC₅₀ of 62 nM for the β₂ receptor. Theβ₁/β₂ functional ratio was found to be >484.

By way of comparison, the values found for arformoterol were 3 nM (β₁),0.041 nM (β₂) and 75.

Stability in Aqueous Buffered Solutions

Solution Preparations: For each test compound, the following solutionsare prepared.

-   -   Solution A is prepared from ˜30 mg of the test compound in 150        mL of 0.005 M citrate buffer, pH 5.0 (˜0.2 mg/mL).    -   Solution B is prepared as follows: approximately 30 mL aliquot        of Solution A is transferred to a separated container and the pH        of the solution is adjusted to pH 3.0 with 1 N HCl (˜0.2 mL).    -   Solution C is prepared as follows: approximately 30 mL aliquot        of Solution A is transferred to a separated container and the pH        of the solution is adjusted to pH ˜8.0 with 1 N NaOH (˜0.2 mL).    -   Note: Because the volume of 1 N HCl or 1 N NaOH used for        adjusting pH was negligible, the concentration of test compound        in Solutions A, B and C were the same.

Storage Scheme

-   -   As soon as the above solutions were prepared, aliquots of each        solution were transferred into 11 vials, of which 9 vials are        stored at −20° C., and one each is stored at 30° C. and 40° C.,        respectively.    -   At each interval listed below, two vials are removed from        −20° C. storage, and stored at 30° C. and 40° C., respectively.    -   The corresponding weeks under the storage condition (30° C. or        40° C.) are shown in the table below.

Week of Vial Removal 0 4 8 10 11 12 Weeks Under Storage Condition 12 8 42 1 0 (30° C. or 40° C.)

On Week 12, the last vial stored at −20° C. is removed and warmed up tothe room temperature, which is the Day 0 Solution.

Sample Analysis: On Week 12, all solutions are assayed by an HPLC methodusing the Day 0 Solution at pH 5 as a standard solution. The testcompounds were assayed by HPLC with UV detection (refer to Table 1 formethod conditions).

TABLE 1 HPLC Method Conditions Parameter Method Detail Column AtlantisdC18, 3 μm 150 × 4.6 mm Mobile Phase A Water/HCOOH 100/0.1, v/v MobilePhase B ACN/Water/HCOOH 30/70/0.1, v/v/v Column Temp 35° C. Sampler Temp 5° C. Injection Volume 5 μL Flow Rate 0.6 mL/min Wavelength PDA 200-350nm Run Time 50 min Time (min) % A % B Gradient 0 100 0 Table 8 100 0 1570 30 45 5 95 46 100 0 50 100 0

TABLE 2 Stability of SEP-229115 in Aqueous Solution (% of InitialConcentration (0.19847 mg/mL) in pH 5.0) Condition pH 3.0 pH 5.0 ~pH 8Time(wk) 30° C. 40° C. 30° C. 40° C. 30° C. 40° C. 0 98.31 98.31 100.20100.20 97.78 97.78 1 99.95 98.87 99.68 100.40 69.33 30.18 2 99.12 100.33101.08 101.07 50.77 8.85 4 100.91 105.71 101.59 102.09 27.72 0.52 8101.16 106.72 105.28 104.88 6.96 0 12  101.54 106.51 106.61 106.74 1.240

Conclusion: At pH 3 or 5, the compound of Example 1 was found to be verystable for at least 12 weeks when stored at 30° C.

1. A compound of formula (I)

or a pharmaceutically acceptable salt thereof.
 2. A mixture of isomersof methyl[2-hydroxy-5-[1-hydroxy-2-[[(2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-phenyl]carbamate, wherein the mixture comprises at least 90% by

weight of the isomer.
 3. A compound as claimed in claim 1 or claim 2,which is in the form of a D-tartrate salt.
 4. A process for thepreparation of a compound as defined in any one of claims 1 to 3, whichcomprises reacting a compound of general formula (II)

in which P¹ represents a hydrogen atom or a hydroxyl protecting group,with a compound of general formula (III)

in which P² represents a hydrogen atom or a hydroxyl protecting groupand P³ represents a benzylic amine protecting group, to afford acompound of general formula (IV)

or a salt thereof, followed by removing any protecting groups P¹, P² andP³ and, if desired, forming a pharmaceutically acceptable acid additionsalt.
 5. A compound of general formula (IV)

or a salt thereof, in which P¹ and P² each represents a hydrogen atom ora hydroxyl protecting group, and P³ represents a hydrogen atom or abenzylic amine protecting group, provided that at least one of P¹, P²and P³ represents a protecting group.
 6. A pharmaceutical composition,which comprises a compound as claimed in any one of claims 1 to 3 and apharmaceutically acceptable carrier.
 7. A pharmaceutical composition asclaimed in claim 6, which further comprises a steroid.
 8. Apharmaceutical composition as claimed in claim 7, in which the steroidis beclomethasone, triamcinolone, funisolide, mometasone, budesonide orfluticasone.
 9. A pharmaceutical composition as claimed in any one ofclaims 6 to 8, which further comprises a muscarinic receptor antagonist.10. A pharmaceutical composition as claimed in claim 9, in which themuscarinic receptor antagonist is ipratropium or tiatropium.
 11. Apharmaceutical composition as claimed in any one of claims 6 to 10 whichfurther comprises an anticholinergic.
 12. A pharmaceutical compositionas claimed in claim 11, in which the anticholinergic is glycopyrralate.13. A pharmaceutical composition as claimed in any one of claims 6 to 12which further comprises a mucolytic.
 14. A pharmaceutical composition asclaimed in claim 13, in which the mucolytic is cromoglycate,acetylcysteine, arginine, or 2-mercaptoethanesulphonate.
 15. Apharmaceutical composition as claimed in any one of claims 6 to 14 whichfurther comprises an anti-inflammatory.
 16. A pharmaceutical compositionas claimed in claim 15, in which the anti-inflammatory agent is aninhibitor of tumor necrosis factor alpha (TNFα) or dipeptidyl peptidaseIV, and/or antibodies to pro-inflammatory interleukins such as IL4 andIL13.
 17. A method for treating bronchoconstriction associated with areversible obstructive airways disease, which comprises administering toa patient in need of treatment, an effective amount of a compound asclaimed in claim 1.